Korean Red Ginseng Suppresses Metastasis of Human Hepatoma SK-Hep1 Cells by Inhibiting Matrix Metalloproteinase-2/-9 and Urokinase Plasminogen Activator

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Ho Y-L, Li K-C, Chao W, et al. Evidence-Based Complementary and Alternative Medicine. Volume 2012 (2012), doi:10.1155/2012/965846

Korean red ginseng and ginsenosides have been claimed to possess wide spectrum of medicinal effects, of which anticancer effect is one. The present study was undertaken to investigate the antimetastatic effect of Korean red ginseng on human hepatoma as well as possible mechanisms. The inhibitory effect of the water extract of Korean red ginseng (WKRG) on the invasion and motility of SK-Hep1 cells was evaluated by the Boyden chamber assay in vitro. Without causing cytotoxicity, WKRG exerted a dose-dependent inhibitory effect on the invasion and motility, but not adhesion, of highly metastatic SK-Hep1 cells. Zymography analyses revealed significant downregulating effects on MMP-2, MMP-9, and uPA activities in SK-Hep1 cells. Western blot analyses also showed that WKRG treatment caused dose-dependent decreases in MMP-2 and MMP-9 protein expressions. Moreover, WKRG increased the levels of TIMP-1, TIMP-2, and PAI-1. The present study not only demonstrated that invasion and motility of cancer cells were inhibited by WKRG, but also indicated that such effects were likely associated with the decrease in MMP-2/-9 and uPA expressions of SK-Hep1 cells.

In this study, we explored the anti-metastatic effect of Korean red ginseng in human hepatoma SK-Hep1 cells. Analytical data received from the Korean Ginseng Corporation showed that there was a great variation in ginsenoside contents between WKRG and EKRG, most likely due to the different extracting processes. Although the difference in the effect of these two extracts on cell viability was not prominent, there was a great variation in their inhibitory effects on MMP-2/-9 activities. WKRG inhibited MMP-2/-9 activities in a concentration-dependent manner, while EKRG had no significant inhibition on MMP-2/-9 activities at all tested concentrations. Based on the above observation, WKRG was selected to do the follow-up experiments in this paper. We found that WKRG also diminished uPA activity of SK-Hep1 cells. Moreover, WKRG significantly inhibited the invasion (assessed using the transwell assay) and migration (examined by both transwell and wound-healing assays) of SK-Hep1 cells. We further demonstrated that WKRG notably inhibited the protein expressions of MMP-2 and MMP-9 and increased the protein levels of TIMP-1, TIMP-2 and PAI-1. These results indicated that antimetastatic effect of WKRG was related to the inhibition of enzymatically degradative processes of tumor metastasis. To our knowledge, this is the first study that attempted to explore biochemical mechanisms underlying WKRG’s inhibitory effect on the metastasis of SK-Hep1 cells.Panax ginseng is an herb frequently used in traditional oriental medicine for its wide spectrum of medicinal effects such as tonic, immunomodulatory, anticancer, adaptogenic, antiaging, antioxidant, and neuroprotective effects [10].

The major active components of ginseng are ginsenosides. More than forty ginsenosides have, so far, been isolated and each ginsenoside possesses different pharmacological effects [11]. Commercially available Korean ginseng products are classified into fresh ginseng, white ginseng, and red ginseng. White ginseng is fresh ginseng which has been air-dried, while red ginseng is obtained by heating. Although processed differently, both red and white ginseng products are manufactured from 6-year-old fresh ginseng roots; nevertheless Korean red ginseng has higher contents of ginsenosides such as Rh2, Rg3, and Rg5 in comparison to white ginseng [12]. Rh2 is produced from ginsenoside Rg3 through bacterial transformation and belongs to the protopanaxadiol family. Rh2 has attracted considerable attention owing to its potential tumor-inhibitory activity. It constrains cell growth in MCF-7 human breast cancer and SK-Hep1 hepatoma and can induce apoptosis in various cell lines [13]. Ginsenoside Rg3 has been reported to reduce the gelatinolytic activities of MMP-2 and MMP-9 [14]. Other important ginseng saponins with unique chemopreventive actions include Rb2 which may partly contribute to the inhibition of lung tumor metastasis by arresting tumor-associated angiogenesis [15], and Rp1 (a semisynthesized derivative of ginsenoside Rg5) whose anticancer effect is believed to be achieved by strongly inhibiting tumor cell metastasis and viability, presumably through impeding adhesion and vessel formation [16].Cell migration is a complex process involving many types of intracellular and extracellular components and is associated with signaling pathways. Since migration is a critical event in cancer progression and especially in metastasis, the inhibitory effect of WKRG on cell migration was evaluated [4]. We found that WKRG significantly inhibited the migration and invasion of SK-Hep1 human hepatoma cells. Invasion of cancer cells through a coated membrane involves not only ECM degradation, but also the formation of adhesive interactions between cells and the matrix. Therefore, the cell adhesion assay was carried out; however we arrived with the result that WKRG only caused a small reduction in cell adhesion. The slight reduction in adhesion may be associated with the significant decreases in migration and invasion by WKRG treatment.MMPs belong to a family of zinc-dependent endopeptides. They are secreted as inactive proenzymes and are activated by partial proteolytic cleavage. MMP-2, MMP-9 and dominant MMPs are released by most endothelial cells and appear to play important roles in the degradation of type VI collage, a major constituent of basement membrane, in cancer invasion and metastasis [17].

In this study, we observed up to 62.6% and 81.5% downregulations of MMP-2 and MMP-9 activities by WKRG as compared with the control. Although these are not direct inhibitions of enzymatic activity, they seem to be enough to decrease cancer metastasis, based on other studies [18].The expression of uPA has been suggested to play a critical role in local fibrin deposition/dissolution [17]. Conversion of plasminogen into active plasmin by plasminogen activators, such as uPA, is primary for fibrinolysis to occur. Plasmin degrades fibrin and prevents its extracellular deposition. WKRG caused a reduction in cell surface plasmin activity as evidenced by the uPA activity assay. The importance of fibrinolytic system in wound healing has been demonstrated in plasminogen-deficient mouse models, where healing is weakened primarily due to impaired fibrinolysis, a consequence of insufficient plasmin generation [19]. Furthermore, uPA plays prominent roles in cellular migration and is vital during the initial phases of wound healing [20]. We used the wound healing assay in vitro to observe the effect of WKRG on cellular migration. WKRG potently retarded the migration of cells towards the wounded area. The results of this study demonstrated that WKRG inhibited fibrinolysis and cell migration which are vital during the early phase of wound healing.To further explore the mechanisms underlying the anti-metastatic effect of WKRG, we detected the alteration in levels of several proteins by lysing SK-Hep1 cells after they had been incubated in the presence or absence of WKRG, including MMP-2, MMP-9, TIMP-1, TIMP-2, and PAI-1.

There is in vitro evidence that MMPs and TIMPs are critical in determining the invasive potential of proliferating tumor cells. MMP-2 and MMP-9 are necessary for the migration of many normal cell types and tumor cells [21]; on the other hand, TIMPs are believed to play important roles in the inhibition of growth and migration, especially in hepatocellular carcinomas [22]. These documented findings strongly suggest anti-metastatic potential of WKRG, as we have demonstrated that WKRG suppressed MMP-2/-9 and enhanced TIMP-1/-2 expressions in terms of protein levels.PAI-1 is a serine protease inhibitor which inactivates uPA to prevent it from binding to the uPA receptor (uPAR). uPAR is part of the plasminogen activation system which is also involved in the regulation of cell adhesion, migration, and invasion. It can transmit uPA-mediated extracellular signals inside the cell, probably through the association with different types of integrins and ECM components. Thus PAI-1 has the ability to arrest the proteolysis cascade following uPAR activation [23]. Our experiment by Western blot demonstrated that the protein level of PAI-1 was enhanced in a dose-dependent manner by WKRG treatment. In summary of the above results, it is suggested that WKRG’s influences on MMP-2, MMP-9, uPA, PAI-1, TIMP-1, and TIMP-2 expressions may have a synergic suppressive effect on the migration and invasion of tumor cells.In conclusion, we explored the anti-metastatic effects and mechanistic actions of WKRG in human hepatoma SK-Hep1 cells. It was found that WKRG significantly inhibited the invasion and migration of SK-Hep1 cells. Then, we carried forward the study by showing that WKRG notably inhibited the expressions of MMP-2, MMP-9, and uPA and elevated the protein levels of PAI-1, TIMP-1, and TIMP-2. These results not only evidenced anti-metastatic effect of WKRG but also showed that such effect was associated with the inhibition of enzymatically degradative processes of tumor metastasis. The present study suggests that Korean red ginseng may be developed into a promising agent for cancer therapy. As to which ginsenosides are predominantly responsible for the anti-metastatic effect of Korean red ginseng remain primarily unclear and would require further studies.

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